ISOLATION AND CHARACTERIZATION OF ONE SOLUBLE AND 2 MEMBRANE-ASSOCIATED FORMS OF PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE-C FROM HUMAN-PLATELETS
ISOLATION AND CHARACTERIZATION OF ONE SOLUBLE AND 2 MEMBRANE-ASSOCIATED FORMS OF PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE-C FROM HUMAN-PLATELETS
复制标题
DOI：
10.1021/bi00440a043
复制
发表时间：
1989-07-11
期刊：
影响因子：
2.9
通讯作者：
FISHER, GJ
中科院分区：
文献类型：
--
作者：
BALDASSARE, JJ;HENDERSON, PA;FISHER, GJ
两种形式（mPLC-I，mPLC-II）的磷酸肌醇特异性磷脂酶C已被纯化，1494和1635倍，分别从人血小板的质膜。纯化的mPLC-I和mPLC-II通过凝胶过滤和十二烷基硫酸钠-聚丙烯酰胺凝胶的估计分子量分别为69 000和63 000。两种胞质形式（PLC-I和PLC-II）的磷酸肌醇特异性磷脂酶C也解决了苯基-琼脂糖柱。通过在Fast Q-Sepharose、磷酸纤维素、肝素-琼脂糖、苯基-Sepharose、Superose 12、DEAE-5 PW和羟基磷灰石上进行层析，将过期血小板中存在的主要胞质形式PLC-II纯化至均一。纯化的PLC-II在十二烷基硫酸钠-聚丙烯酰胺凝胶上的分子量为57 000。mPLC-I、mPLC-II和PLC-II水解PI和PIP 2。对于所有三种形式的PLC，PIP 2水解的Vmax相似，并且比PI水解的Vmax大约5倍。PIP 2水解的Km对于三种酶也是相似的。相比之下，PLC-II对PI水解的Km比mPLC-I和mPLC-II低10倍。此外，针对PLC-II制备的抗体不与mPLC-I或mPLC-II交叉反应。这些数据表明，血小板含有膜相关的磷酸肌醇特异性磷脂酶C，不同于至少一种胞质形式（PLC-II）的酶。
Two forms (mPLC-I, mPLC-II) of phosphoinositide-specific phospholipase C have been purified, 1494- and 1635-fold, respectively, from plasma membranes of human platelets. Purified mPLC-I and mPLC-II had estimated molecular weights by gel filtration and sodium dodecyl sulfate-polyacrylamide gels of 69 000 and 63 000, respectively. Two cytosolic forms (PLC-I and PLC-II) of phosphoinositide-specific phospholipase C were also resolved on a phenyl-Sepharose column. The major cytosolic form present in outdated platelets, PLC-II, was purified to homogeneity by chromatography on Fast Q-Sepharose, cellulose phosphate, heparin-agarose, phenyl-Sepharose, Superose 12, DEAE-5PW, and hydroxylapatite. Purified PLC-II had a molecular weight of 57 000 on sodium dodecyl sulfate-polyacrylamide gels. mPLC-I, mPLC-II, and PLC-II hydrolyzed both PI and PIP2. The Vmax for PIP2 hydrolysis was similar for all three forms of PLC and was approximately 5-fold greater than for PI hydrolysis. The Km for PIP2 hydrolysis was also similar for the three enzymes. In contrast, the Km for PI hydrolysis by PLC-II was 10-fold lowr than by mPLC-I and mPLC-II. In addition, antibody prepared against PLC-II did not cross-react with either mPLC-I or mPLC-II. These data indicate that platelets contain membrane-associated phosphoinositide-specific phospholipases C that distinct from at least one cytosolic form (PLC-II) of the enzyme.